Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Choline is a precursor of acetylcholine, phosphatidylcholine, and the methyl-donor betaine. Reports indicate that supplementation with rumen-protected choline improves postpartum reproductive function of dairy cows. The objective was to determine whether addition of choline to culture medium of in vitro-produced embryos alters the phenotype of the resultant blastocysts. Treatments were choline chloride (ChCl; 0.004, 1.3, 1.8, and 6.37 mM) and phosphatidylcholine (1.3 mM). Treatment with 0.004 mM ChCl improved development to the blastocyst stage, increased blastocyst cell number, and increased the percentage of blastocysts that were hatching or hatched. Development was not affected by higher concentrations of ChCl but was reduced by 1.3 mM phosphatidylcholine. Treatment of embryos with 1.3 mM ChCl (but not other concentrations) increased expression in blastocysts of 11 of 165 genes examined (AMOT, NANOG, HDAC8, HNF4A, STAT1, MBNL3, SOX2, STAT3, KDM2B, SAV1, and GPAM) and decreased expression of one gene (ASS1). Treatment with 1.3 mM ChCl decreased global DNA methylation at d 3.5 of development and increased DNA methylation at d 7.5 in blastocysts. Treatment with 1.8 mM ChCl also increased methylation in blastocysts. In conclusion, addition of choline to the culture medium alters the phenotype of preimplantation bovine embryos produced in vitro. Choline chloride can act in a concentration-dependent manner to alter development, expression of specific genes, and DNA methylation.

The objective was to identify the transcriptomic profile of in vitro-derived embryos with high competence to establish and maintain gestation. Embryos produced with X-sorted sperm were cultured from day 5 to day 7 in serum-free medium containing 10 ng/ml recombinant bovine colony-stimulating factor 2 (CSF2) or vehicle. The CSF2 was administered because this molecule can increase blastocyst competence for survival after embryo transfer. Blastocysts were harvested on day 7 of culture and manually bisected. One demi-embryo from a single blastocyst was transferred into a synchronized recipient and the other half was used for RNA-seq analysis. Using P < 0.01 and a fold change >2-fold or <0.5 fold as cutoffs, there were 617 differentially expressed genes (DEG) between embryos that survived to day 30 of gestation vs those that did not, 470 DEG between embryos that survived to day 60 and those that did not, 432 DEG between embryos that maintained pregnancy from day 30 to day 60 vs those where pregnancy failed after day 30, and 635 DEG regulated by CSF2. Pathways and ontologies in which DEG were overrepresented included many related to cellular responses to stress and cell survival. It was concluded that gene expression in the blastocyst is different between embryos that are competent to establish and maintain pregnancy vs those that are not. The relationship between expression of genes related to cell stress and subsequent embryonic survival probably reflects cellular perturbations caused by embryonic development taking place in the artificial environment associated with cell culture.

Understanding the signalling pathways involved with derivation of embryonic stem cells could enhance our understanding of pluripotency in pre-implantation embryos. Recently, the small molecule IWR-1 has been shown to promote derivation of mouse epiblast stem cells and pluripotent bovine and porcine embryonic stem cells (ESC). IWR-1 blocks WNT signalling mediated by β-catenin-targeted gene expression through stabilisation of Axin2, a member of the destruction complex that induces β-catenin degradation. Here, we evaluated whether dickkopf WNT signalling pathway inhibitor 1 (DKK1) can replace IWR-1 for establishment of bovine pluripotent ESC. If so, it is likely that the actions of IWR-1 to promote pluripotency involve inhibition of WNT signalling. Treatment of bovine embryos with 100ngmL−1 recombinant human DKK1 beginning at Day 5 of development decreased (P=0.02) immunofluorescent labelling of β-catenin in the resulting blastocysts (n=41-45/group), indicating that bovine embryos are responsive to DKK1 treatment. For ESC derivation, blastocysts were plated on top of feeder cells and cultured in ESC medium supplemented with 2.5 µM IWR-1 (n=21), 100ngmL−1 DKK1 (n=34), or vehicle (n=23). Cells were passaged every 5 to 7 days in their respective treatment medium. Seven days after plating, 57.9±14.7% of blastocysts in IWR-1 ESC medium developed outgrowth, which was lower (P=0.02) than the proportion of blastocysts with outgrowth in DKK1 medium (92.4±5.2%) or vehicle (81.9±10.0%). Outgrowth size did not differ among treatments. Labelling with CDX2 indicated that the majority of cells in outgrowths were trophectoderm cells. Thus, IWR-1 inhibits competence of blastocysts to form trophectoderm outgrowths during derivation of ESC. The percent of blastocysts from which cell lines were derived after 4 passages were 48% (10/21) for IWR-1, 41% (14/34) for DKK1, and 48% (11/23) for vehicle. Immunolabelling for the pluripotency marker SOX2 showed that only cells grown in IWR-1 medium were positive, whereas most of the cells derived in the other two media were not. Thus, IWR-1 could not be replaced by DKK1 for maintaining pluripotency. Immunoreactive β-catenin was abundantly distributed on the membrane of cells cultured with IWR-1 but not with DKK1 or vehicle-treated cells. Thus, β-catenin distribution to the cell membrane is linked with bovine pluripotency. Overall, results indicate that maintenance of pluripotency by IWR-1 may involve mechanisms other than WNT inhibition, and may be related to the localization of β-catenin to the plasma membrane.

Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus-oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.

Embryokines are molecules secreted by the mother that regulate embryonic development. Among these molecules in cattle are colony stimulating factor 2 (CSF2) and dickkopf-related protein 1 (DKK1). Here, we evaluated actions of CSF2 and DKK1 alone or in combination on characteristics of embryos produced in vitro in the presence of serum. A total of 70 beef cows from 4 farms were subjected to oocyte retrieval on 1 to 4 occasions. Within each farm, donors were randomly allocated to 1 of 4 treatment groups (vehicle, CSF2, DKK1, CSF2 + DKK1). Embryos from a given donor were always exposed to the same treatment. Treatments were added to the culture medium on d 5 after insemination, and blastocyst stage embryos were transferred to recipient females 2 d later. Treatment did not affect the percent of oocytes or cleaved embryos that developed to the blastocyst stage or the percent of recipients that became pregnant after embryo transfer. However, calves derived from embryos treated with DKK1 were smaller at birth, regardless of CSF2 treatment. Results indicate no effects of addition of CSF2 or DKK1 to culture of embryos produced in vitro with serum-containing medium on development to the blastocyst stage or competence to establish pregnancy after transfer to recipients. The fact that embryos cultured with DKK1 resulted in calves with reduced birth weight illustrates the potential ability of this embryokine to program postnatal phenotype. Results support the concept that properties of the offspring can be programmed as early as the preimplantation period.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the pre-implantation embryo, called embryokines, is largely incomplete. To identify possible candidates for this function, an experiment was conducted to assess expression patterns during the first 7 days after ovulation for 92 genes that could be involved in control of development. Included were genes for 27 growth factors, 11 cytokines, 22 interleukins, 3 hormones, 19 WNT ligands, and 9 WNT regulatory molecules. Cows were slaughtered at Days 0, 3, 5, and 7 relative to predicted ovulation. Reproductive tracts were obtained and transversal sections from the isthmus of oviducts ipsi- and contralateral to the corpus luteum were harvested for gene expression analysis. Abundance of specific mRNA molecules was determined using the NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA). Data were normalized against 6 housekeeping genes (ACTB, ERK1, GAPDH, RPL19, SLC30A6, SUZ12) and internal positive controls. Genes were considered expressed if the number of reads was greater than 2 standard deviations above the mean of negative controls. Data were analysed by ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) with day, side, and day × side as fixed effects, and cow as random effect. Side did not have any significant effect so data were analysed without side and day × side in the model. In contrast to what was observed earlier for endometrium (P. Tribulo and P. J. Hansen, unpublished data), we found no difference in gene expression between oviducts ipsi- and contralateral to the corpus luteum. Overall, there was wide variation in the magnitude of gene expression. Among the 20 most expressed genes, average reads varied from 164 to over 10 726. All genes were detected at Days 0, 3, and 7 but only 67 of the 92 genes were expressed at Day 5. The 10 highest-expressed genes were CTGF, CXCL3, CXCL10, CXCL12, GRO1, IGF2, IK, SFRP1, WNT5A, and WNT6. Of these, CTGF, CXCL12, IGF2, IK, HDGF, WNT5A, and CXCL3 were within the 10 highest expressed at all days (P. Tribulo and P. J. Hansen, unpublished data). There were only 6 genes whose expression was significantly affected by day. Expression was highest at oestrus (VEGFA), Day 5 (GRO1, SFRP1) or Day 7 (BMP4, IK, WNT16). This experiment identifies some potential maternal regulators of embryonic development. Expression of most of these putative embryokine genes did not vary with stage of the oestrous cycle, suggesting that expression is either not under endocrine control or varies between cell types within the oviduct. Further studies are needed to determine the effect of these maternally secreted molecules on embryonic development. Study was supported by the National Institutes of Health (HD080855).

Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.

In vitro production (IVP) of embryos can disrupt fetal and placental development and increase risk of abnormal fetal growth. Maternal factors play a role in developmental programming of the early embryo. Colony-stimulating factor 2 (CSF2) is present in the oviduct and endometrium and has improved competence of the pre-implantation embryo to establish pregnancy in cattle. The objective was to determine whether CSF2 during embryo culture alters fetal development and alleviates abnormalities associated with IVP. Holstein oocytes were matured and fertilised in vitro with X-sorted semen from a Holstein bull. Putative zygotes were cultured in SOF-BE1 at 5% CO2 and 5% O2 for 5 days and then randomly assigned to receive vehicle (IVP-control) or 10 ng mL–1 CSF2 (IVP-CSF2). Grade I blastocysts were transferred on Day 7 to Holstein recipients that were previously randomised to receive an IVP-control or an IVP-CSF2 embryo. A third group of cows included in the randomization was assigned to be artificially inseminated on Day 0 using the same bull as for IVP (AI). Pregnancy was terminated on Day 85 or 86. Statistical analysis was performed by analysis of variance using the GLM procedure of SAS with contrasts for AI v. (IVP-control+IVP-CSF2) (contrast 1; C1) and IVP-control v. IVP-CSF2 (contrast 2; C2). Results are least squares means ± s.e.M. A total of 23 morphometric measurements of placenta and fetus were made on 9 AI, 12 IVP and 7 CSF2 female singletons. Conceptuses derived by IVP (IVP-control and IVP-CSF2) differed from those derived by AI for 4 characteristics including fetal bodyweight (142.9 ± 4.7, 157.2 ± 4.4, and 162.6 ± 6.1 g for AI, IVP-control and IVP-CSF2, respectively; C1, P = 0.0237), eviscerated weight (102.9 ± 3.4, 113.6 ± 3.2, and 112.2 ± 4.4 g; C1, P = 0.0602), crown-rump length (CRL) (13.7 ± 0.2, 14.0 ± 0.2, and 14.7 ± 0.3 g; C1, P = 0.0434; C2, P = 0.0631) and umbilical cord diameter (0.85 ± 0.08, 1.1 ± 0.08, and 0.91 ± 0.1 cm; P = 0.0519). Note that while IVP-CSF2 conceptuses were generally similar to those for IVP-control, CRL tended to be highest for IVP-CSF2. Also, umbilical cord diameter for IVP-CSF2 was similar to AI and lower than IVP-control. Data from 1 fetus in the IVP-CSF2 group was excluded from analysis because it had a phenotype consistent with large offspring syndrome. Bodyweight (354 g) was 2-fold larger than other fetuses (average = 155 g) and placental weight was 7-fold greater (1505 v. 211 g). In addition, organs were enlarged and severe ascites and hemorrhagic cotyledons were observed. In conclusion, IVP resulted in increased fetal size and umbilical cord diameter without other significant effects on placental morphometry. CSF2 did not alleviate adverse effects of culture on fetal growth, exacerbating effects on CRL, but did reduce effects of IVP on umbilical cord diameter. Gene expression analysis may be useful for further characterisation of effects and elucidation of mechanisms involved. This project was supported by USDA NIFA Grant 2011–67015–30688.

Wingless-related mouse mammary tumour virus (WNT) signalling participates in early embryonic development to maintain pluripotency, controls cell–cell communication, and modulates cell polarization and migration. To gain an understanding of the regulation of WNT signalling during embryonic development, expression patterns of a variety of molecules involved in WNT signal transduction were evaluated. Specific genes were DKK1, an endogenous inhibitor of canonical WNT signalling, the WNT co-receptors LRP5 and LRP6, WNT-responsive transcription factors, LEF1 and TCF7, and two repressors of WNT-regulated genes, the bovine orthologue of GROUCHO (LOC505120) and AES. Embryos were produced in vitro from oocytes obtained from ovaries collected at a local abattoir. Following oocyte maturation, fertilization was performed with sperm pooled from three randomly selected bulls; a different pool of bulls was used for each replicate. Groups of 30 matured oocytes or embryos at the 2-cell [28–32 h post-insemination (hpi)], 3–4 cell (44–48 hpi), 5–8 cell (50–55 hpi), 9–16 cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The zona pellucida was removed with proteinase, RNA was purified, cDNA synthesised using random hexamer primers and real-time qPCR performed. Data analysed were ΔCT values, which were calculated by subtracting the CT value of the geometric mean of the three housekeeping genes (GAPDH, YWHAZ, and SDHA) from the CT value of the sample. The relative transcript abundance was calculated as the 2ΔCT. Data were analysed by least-squares ANOVA using the Proc GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). A total of 5 replicates were analysed for each developmental stage. Results show significant effects of stage of development for each gene that ranged from P = 0.004 for LRP5 to P ≤ 0.0001 for AES, DKK1, LEF, LOC505120, LRP6, and TCF7. In all cases, expression declined as development advanced. Except for AES, lowest expression occurred at the blastocyst stage. Lowest expression for AES was at the morula stage; expression remained low at the blastocyst stage. For two genes, DKK1 and LEF1, there was no detectable expression at the blastocyst stage. The timing of decline in expression varied between genes, first occurring at the 9–16-cell stage (AES, LEF1, and LOC505120) or morula stage (DKK1, LRP5, LRP6, or TCF7). For DKK1, LEF1, and LRP6, there was also a slight increase in expression from the oocyte to two-cell stage. Results suggest that canonical WNT signalling is reduced at the morula and blastocyst stages relative to earlier stages in development. Research was supported by USDA-NIFA 2011-67015-30688.

To understand the role of ovulation-inducing factor (or nerve growth factor) (OIF [NGF]) in bovine seminal plasma, we (1) used an in vivo llama bioassay to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas similar to that of llama seminal plasma when the dose of seminal plasma is adjusted to ovulation-inducing factor content (experiment 1) and (2) determined the effect of bovine seminal plasma on the interval to ovulation and luteal development in heifers (experiment 2). Within species, seminal plasma was pooled (n = 160 bulls, n = 4 llamas), and the volume of seminal plasma used for treatment was adjusted to a total dose of 250 μg of ovulation-inducing factor. In experiment 1, mature female llamas were assigned randomly to four groups and treated intramuscularly with either 10 mL of PBS (negative control, n = 5), 50-μg GnRH (positive control, n = 5), 6-mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. In experiment 2, beef heifers were given a luteolytic dose of prostaglandin followed by 25-mg porcine LH (pLH) 12 hours later to induce ovulation. Heifers were assigned randomly to three groups and given 12 mL bovine seminal plasma intramuscularly 12 hours after pLH treatment (n = 10), within 4 hours after ovulation (n = 9), or no treatment (control, n = 10). Ovulation was monitored by ultrasonography every 4 hours, and the CL development was monitored daily until the next ovulation. In experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 llamas in the PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P < 0.05). Luteal development was not different among groups. In experiment 2, the interval to ovulation was more synchronous (range: 4 vs. 22 hours; P < 0.0001) in heifers treated with seminal plasma before ovulation compared with the other groups. Luteal development was not different among groups; however, plasma progesterone concentrations tended to be greater in the postovulation treatment group compared with other groups. In summary, results confirmed the presence of bioactive ovulation-inducing factor in bull seminal plasma and supported the hypothesis that bovine and llama seminal plasma have similar ovulatory effects, using a llama bioassay. Treatment with bovine seminal plasma resulted in greater synchrony of ovulation in heifers pretreated with pLH. Plasma progesterone concentration tended to be higher in heifers given bovine seminal plasma within 4 hours after ovulation, suggesting that bovine ovulation-inducing factor is luteotrophic.

Ovulation-inducing factor (OIF) is a protein present in the seminal plasma of several species, including llamas, alpacas, pigs, cattle, sheep, horses and rabbits. In an initial study (Ratto et al. 2006 Theriogenology 66, 1102–1106), bovine seminal plasma induced ovulations in 26% (5/19) of llamas compared with 0% (0/19) in the placebo group, but induced proportionately less than in llamas treated with alpaca or llama seminal plasma (100%). It is important to highlight that treatments were based on volume of seminal plasma; the actual dose of OIF was unknown. In a later study (Tanco et al. 2011 Biol. Reprod. doi:10.1095/biolreprod.111.091876), OIF from llama seminal plasma had a dose-dependent effect on ovulation rate, corpus luteum (CL) diameter and progesterone production in llamas. The present study was designed to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas comparable with that of llama seminal plasma, based on total dose of OIF. Within species, seminal plasma was pooled from 1 to 4 ejaculates per male (n = 145 bulls, n = 4 llamas). The concentration of OIF in the pooled seminal plasma was measured by radioimmunoassay and the volume of seminal plasma used for treatment was adjusted to reach a total dose of 250 μg of OIF. Mature female llamas were assigned randomly to 4 groups and given a single intramuscular dose of 10 mL of PBS (negative control, n = 5), 50 μg of gonadotropin-releasing hormone (GnRH; positive control, n = 5), 6 mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. The incidence of ovulation was compared among groups by Fisher's exact test. Nonserial data (i.e. follicle size at treatment, maximum CL diameter, day of maximum CL diameter and first day of CL detection) were compared among groups by ANOVA. The diameter of the preovulatory follicle at treatment did not differ among groups (P = 0.10). The incidence of ovulation was 0/5, 4/5, 3/6 and 4/6 in the groups treated with PBS, GnRH, llama seminal plasma and bovine seminal plasma, respectively (P < 0.05). The incidence of ovulation did not differ among llamas treated with GnRH, llama seminal plasma, or bovine seminal plasma. Among the treatments that elicited ovulation, neither the maximum CL diameter nor the day of maximum CL diameter differed (P = 0.30 and P = 0.24, respectively). In addition, no difference was detected in the day of first detection of the CL (P = 0.25). Results document the bioactivity of OIF in the bovine seminal plasma of Bos taurus. These findings further support the notion that OIF is highly conserved among mammals and that seminal plasma exerts its effect in an OIF dose-related manner. This research was supported by the Natural Sciences and Engineering Research Council of Canada.

Two experiments were designed to evaluate the superovulatory response of Brangus and Bonsmara donor cows to different dosages of Folltropin®-V (Bioniche Animal Health Inc., Belleville, Ontario, Canada) given by a single i.m. injection or twice-daily i.m. injections. In Experiment 1, Brangus cows (n = 12) were superstimulated by 6 treatments (2 × 3 factorial) in a crossover design (i.e. all cows received the 6 treatments and all treatments were represented on each day). On Day 0, cows received 5 mg of estradiol-17β plus 50 mg of progesterone and a Cue-Mate® (Bioniche Animal Health Inc.). On Day 4, cows were superstimulated with 300, 260, or 200 mg of NIH-FSH-P1 Folltropin®-V (Bioniche Animal Health Inc.) in twice-daily decreasing doses over 4 days or diluted in a slow release formulation (SRF; Bioniche Animal Health) and given in a single i.m. injection. The single injection was prepared by diluting the Folltropin®-V lyophilized powder in 1 mL of saline followed by mixing with 9 mL of the SRF in the syringe immediately before administration. In the am and pm of Day 6, all cows received PGF2, and Cue-Mates® were removed in the pm. Cows received 12.5 mg of porcine LH (Lutropin®-V; Bioniche Animal Health Inc.) in the am of Day 8 and were inseminated 12 and 24 h later. Ova/embryos were collected on Day 15 and data were analyzed by ANOVA. There was no effect of treatment (i.e. single v. twice-daily injections; P > 0.2) nor a treatment by dosage interaction (P < 0.6) on the mean (± SEM) number of total ova/embryos or transferable embryos (13.1 ± 1.9 and 7.5 ± 1.2 v. 15.5 ± 1.7 and 7.6 ± 1.0 for single v. twice-daily injections, respectively). The total number of ova/embryos did not differ among Folltropin®-V dosages (15.0 ± 2.3, 15.7 ± 2.0, and 12.1 ± 2.5 for 300, 260, and 200 mg, respectively; P > 0.4). However, the number of transferable embryos tended (P < 0.09) to be higher in donors receiving 260 mg (9.5 ± 1.6) than 200 mg (5.2 ± 0.8), with 300 mg (7.9 ± 1.5) intermediate. In Experiment 2, Bonsmara cows (n = 16) were superstimulated by 4 treatments (2 × 2 factorial) in a crossover design similarly to Experiment 1, except that 2 dosages of Folltropin®-V (200 and 300 mg) were evaluated. There were no significant effects of dosage of Folltropin®-V (P > 0.9), treatment (P > 0.3), or interaction (P < 0.4) on embryo production. The total number of ova/embryos and transferable embryos were 11.9 ± 2.0 and 7.2 ± 1.1 v. 11.1 ± 1.1 and 7.6 ± 0.7 for single and twice-daily injections, respectively, and 11.9 ± 1.9 and 7.6 ± 1.0 v. 11.1 ± 1.3 and 7.2 ± 0.8 for 300 and 200 mg of Folltropin®-V, respectively. Superstimulation of Brangus and Bonsmara cows with a single i.m. injection of Folltropin®-V diluted in a SRF resulted in comparable embryo production to twice-daily administration of Folltropin®-V over 4 days. While 260 mg seems to be the most appropriate dosage for Brangus donors, 200 mg seems to be adequate for Bonsmara donors.